Association of LAMA1 Single-Nucleotide Polymorphisms with Risk of Esophageal Squamous Cell Carcinoma among the Eastern Chinese Population

Introduction LAMA1, also known as laminin subunit α1, is a member of the laminin family, which is widely reported to be a key basement membrane molecule that affects various biological activities and is associated with many kinds of diseases. We aimed to investigate the association between LAMA1single-nucleotide polymorphisms and the occurrence and progression of esophageal squamous cell carcinoma in the Chinese population. Method 2,186 participants were collected retrospectively between October 2008 and January 2017, including 1,043 ESCC patients and 1,143 noncancer patients. A 2 mL blood sample was obtained intravenously for the LDR for SNP analysis. The 6 SNP loci of LAMA1 were selected and examined. We analyzed the association of several genetic models of 6 LAMA1 SNP loci, sex, age, smoking and drinking status, and the occurrence of esophageal squamous cell carcinoma. Results In the rs62081531 G > A locus, genotype GA was a protective factor for ESCC compared with GG (OR: 0.830, P=0.046), especially among the younger and nondrinkers. At rs607230 T > C, genotype TC was linked with a lower risk of ESCC compared with TT. (OR: 0.613, P=0.034). Haplotype Frequencies revealed that Ars62081531Grs621993Ars539713Trs566655Ars73938538Crs607230 (OR: 0.803, P=0.028) and Grs62081531Grs621993Ars539713Trs566655Crs73938538Crs607230 (OR: 0.679, P=0.010) were strongly associated with lower susceptibility of ESCC. Conclusion The LAMA1 rs62081531, rs539713, rs566655, and rs607230 polymorphisms were demonstrated to be related to susceptibility to ESCC in the Chinese population. LAMA1 SNPs may have a significant impact on the occurrence of esophageal cancer and may serve as potential diagnostic biomarkers.


Introduction
Esophageal cancer is a prevalent malignant tumor that has a high rate of morbidity and mortality worldwide. According to the Global Cancer Statistics 2020, esophageal cancer is more prevalent in East Asia, particularly in China, as well as West Asia and Africa [1]. Tere was a clear correlation between the pathological type of esophageal cancer and its geographic distribution. Squamous cell carcinoma is the most common type of esophageal cancer in developing countries. China has a high prevalence of esophageal squamous cell carcinoma (ESCC) of up to 90%. A variety of pathogenic factors may lead to esophageal squamous cell carcinoma, including smoking, drinking, eating habits, and viral infections [2,3]. However, not everyone exposed to these risk factors develops esophageal cancer, suggesting that genetic susceptibility, particularly single nucleotide polymorphisms (SNPs), plays a signifcant role in the development of esophageal squamous cell carcinoma.
LAMA1 is also known as laminin subunit α1. Laminins are a family of glycoproteins found in the extracellular matrix that comprises the basement membrane [4,5]. Laminins have a heterotrimeric structure composed of an α, β, and c chain [6]. Numerous biological processes are known to be directed by them, including cell adhesion, mitogenesis, diferentiation, and metastasis, all of which contribute to carcinogenesis [7][8][9]. Tissue distribution of LAMA1 occurred mainly in early epithelial development and some adult epithelia. Recent reports have shown that laminin-1 acts as an efcient attachment protein for a large variety of cultured cell types in vitro [10]. Mutations in the LAMA1 gene result in a defciency of the laminin α1 chain, which may lead to tumorigenesis and progression [11]. Recent research indicates that LAMA1 mutations or overexpression are linked with the occurrence and development of various malignant tumors, including colon cancer, pancreatic cancer, and ovarian cancer [12][13][14][15].
However, the relationship between the LAMA1singlenucleotide polymorphism and ESCC remains unclear. Trough multicenter large-sample case-control research, we aim to thoroughly investigate the association between LAMA1 single-nucleotide polymorphisms and the incidence and progression of esophageal cancer.

Patients and Study
Design. Between October 2008 and January 2017, 2,186 participants were collected from the Afliated People's Hospital and the Afliated Hospital of Jiangsu University (Zhenjiang, China). Totally, 1,043 cases of esophageal cancer were diagnosed and histologically confrmed as squamous cell carcinoma by two pathologists independently. Patients with a history of any other types of cancer or with metastasis or those who had received neoadjuvant therapy were excluded. Around the same time, 1,143 noncancer patients from both hospitals were enrolled, with a frequency matching by age (±5 years) and gender, and the majority of them were admitted for trauma.
For 1,043 patients and 1,143 negative controls, baseline information such as age, gender, and other ESCC-related risk factors, such as smoking and drinking, were gathered through a questionnaire. A total of 1,143 control individuals and all case subjects provided feedback. Each participant had a 2 mL blood sample obtained intravenously for analysis.
Te protocol adhered to the Declaration of Helsinki on the ethical conduct of research involving human/animal subjects and was approved by the Ethics Committee of Jiangsu University (Zhenjiang, China). All participants signed an informed consent form before recruitment.

Genomic DNA Extraction and Single-Nucleotide
Polymorphism Analysis. Te QIAamp DNA Blood Mini Kit was applied to amplify genomic DNA isolated from peripheral blood using PCR (Qiagen, Berlin, Germany). Samples were genotyped further using the ligation detection reaction (LDR) approach (supported by Genesky Biotechnology Inc., Shanghai, China). Six LAMA1 SNP loci (rs62081531, rs621993, rs539713, rs566655, rs73938538, and rs607230) were selected and analyzed. As a methodology of quality control, the analysis is repeated on 10% of randomly selected samples. In a preliminary study, we performed a linkage disequilibrium analysis on the 1000Genomes database, identifed SNP loci with correlations, and further explored tag SNPs.

Surgical and Histological Evaluation.
All patients underwent esophagectomy by qualifed surgeons. Following the procedure, surgical specimens will be fxed to a cork and immersed in 10% formalin. All these patients' specimens were systematically reevaluated by experienced pathologists specialized in thoracic oncology and restaged under the 8th edition AJCC/UICC staging of cancers of the esophagus and esophagogastric junction. Te histopathological examination includes tumor size, grade of diferentiation, the margin of resection, and lymph node status.

Statistical Analysis.
Statistical analyses were performed using SPSS version 26 (IBM, Chicago, IL) and R software (Version 4.0.3, R Foundation for Statistical Computing, Vienna, Austria). Te baseline characteristics were summarized using the R software package "tableone". Clinical characteristics were compared using Fisher's exact test or the χ 2 test for categorical variables and the Student's t-test for comparing continuous variables. For the genetic model (A as a major allele and B as a minor allele), (a) dominant model: allele B increases risk; (b) Recessive model: two copies of minor allele B are required for increased risk; (c) additive model: r-fold increased risk for AB and 2r increased risk for BB; (d) multiplicative model: r-fold increased risk for AB and r2 increased risk for BB [16]. Two-tailed p value < 0.05 is considered as statistical signifcance, whereas p values between 0.05 and 0.10 are considered borderline statistically signifcant. Te crude odd ratio (OR) and its corresponding 95% confdence interval (CI) are calculated according to genotypes between the two groups. In most cases, the risk is compared using the parametric test. When the sample size in the group is small, we use the nonparametric test. For stratifed analyses, we included age, gender, smoking and drinking status, and the SNP model in the analysis, resulting in an adjusted OR for the SNP model. Te adjusted OR and its corresponding CI are calculated by logistic regression analysis, and the hierarchical analysis is carried out. Demographic data, including age, sex, alcohol, and smoking, were covariates. Te genotype was a dummy variable, and the group was a dependent variable. Te SHEsis online platform [17] was utilized to conduct linkage disequilibrium studies and visualize the results by using R 4.0.3 and the R software packages "LDheatmap" and "genetics," and SHEsis was also used to conduct haplotype frequency analyses [18].

Results
Between October 2008 and January 2017, we included 1,043 patients and 1,143 negative controls. Tere was no statistically signifcant diference in median age between the case and control groups [63.00 (59.00, 68.00) versus 63.00 (54.00, 70.00), p � 0.257]. Women accounted for 27.3% of the case group and 27.6% of the control group (P � 0.941). Te proportion of smoking and drinking in ESCC patients was higher than that in the control group (43.5% vs. 29.7%, P < 0.001, 31.5% vs. 16.3%, P < 0.001). Detailed clinicopathological information was described in Table 1.
Te brief information on the six genotyped SNPs of LAMA1 is shown in Table 2. All SNP genotyping experiments were successful at a rate greater than 95%. Te control group's minor allele frequencies (MAF) were similar to those found in East Asian groups in the 1000 Genomes and gnomAD-Genomes databases. Te Hardy-Weinberg equilibrium (HWE) test revealed that all six SNPs in the control group had p values greater than 0.05, indicating that the control group was genetically equilibrium. Linkage disequilibrium of 6 SNP loci of LAMA1 is shown in Figure 1 and the coefcient of linkage disequilibrium and correlation coefcient test are described in Tables S7a and S7b. Te association of 6 SNPs in LAMA1 with esophageal squamous cell carcinoma is shown in Table 3. In the rs62081531 locus, G/A was a protective factor for ESCC compared with G/G (OR: 0.820, 95% CI: 0.677-0.993, P � 0.042). We can also fnd in the dominant model that the minor allele A is a protective factor for ESCC (OR: 0.830, 95% CI: 0.691-0.997, P � 0.046). At rs607230, T/C was a protective factor for ESCC compared with T/T (OR: 0.613, 95% CI: 0.389-0.965, P � 0.034).

Discussion
Trough our multicenter large-samplecase-control study, we found that rs62081531 locus G > A and rs607230 locus T > C of LAMA1 were independent protective factors for esophageal squamous cell carcinoma, especially in those younger than 65 and nondrinkers. Haplotype frequency analysis revealed that A rs62081531 G rs621993 A rs539713 T rs566655 A rs73938538 C rs607230 and G rs62081531 G rs621993 A rs539713 T rs566655 C rs73938538 C rs607230 were associated with less susceptibility to ESCC among the Chinese population. To our knowledge, this is the frst report on LAMA1 single-nucleotide polymorphisms and susceptibility to esophageal squamous cell carcinoma based on large-scale multicenter clinical research. LAMA1 (laminin subunit α1) is a part of laminin, a glycoprotein found in the extracellular matrix that constitutes the basement membrane, and has been shown to be involved in the occurrence and development of various diseases [11]. Tissue distribution of laminin subunit α1 is mainly in early epithelial development and some adult epithelia [8]. In terms of carcinogenesis, laminin plays an essential role in cell adhesion, mitosis, diferentiation, and even metastasis [19]. Laminin is a fundamental functional component of the basement membrane of several tissues, including the endothelium of the vessel wall, and diferent isoforms may contribute to vascular homeostasis [20]. Te α1 subunit of laminin is typically confned to capillary walls and is expressed in the basal layer of capillaries in the central nervous system [21]. A recent study demonstrates that laminin-1 functions as a chemoattractant for both stromal and vascular cells, as well as in epithelial/stromal cell interactions for the structure of the basement membrane and segregation of integrins, hence signaling the proliferation of epithelial cells [13]. Similarly, in colorectal cancer, Wu et al. reported novel driver mutations occurring during adenoma and cancer evolution by single-cellwhole-exome sequencing (scWES), with LAMA1 (PI3K-Akt signaling pathway) being one of the most critical pathways for CRC evolution [22]. Likewise, Gudjonsson and coworkers revealed that laminin-1 plays a vital role in the replacement of myoepithelial cells in polarity reversal in breast cancer [23]. LAMA1 (laminin α1) mutations are highly related to retinal avascularity and neovascularization in nontumor felds, such as the Poretti-Boltshauser syndrome [24]. Regardless of tumor or other nontumor diseases, LAMA1 is essential for vascular homeostasis and the basal layer of blood vessels.    Journal of Oncology Interestingly, Velling et al. found that none of the colon cancer cell lines synthesized the laminin α1 protein, and they suggested that mutations in the LAMA1 gene may underlie the lack of laminin α1 chains observed in some colon cancers [25]. In our study, LAMA1 mutation showed protective factors in both rs62081531 G > A and rs607230 T > C, and basic research also found that LAMA1 defciency could inhibit the proliferation and invasion of esophageal cancer [26,27]. Clearly, not all LAMA1 SNPS are protective factors. People younger than 65 years of age indicate that the mutation at rs566655 T > G increases the risk of esophageal cancer, which may be correlated to the function of a certain SNP. However, there are few reports on LAMA1 and esophageal cancer. Most of the research is limited to the genetic function of LAMA1. Meng and colleagues found that laminin α1 (LAMA1) is highly expressed in ESCC tissue and mediates the FAK-PI3K-Akt signaling pathway [27]. Zhou et al. revealed that LAMA1 was signifcantly upregulated in ESCC tissues and positively correlated with an aggressive oncogenic phenotype [26]. Nevertheless, the relationship between LAMA1 SNP and disease in malignancies has not been demonstrated. Previous research has focused chiefy on nontumor studies such as chronic disease or degenerative disease. Zhao et al. showed that rs2089760 T > G, which is located in the LAMA1 promoter region, may be associated with myopia in Chinese populations [28]. Similarly, the LAMA1 rs2089760 G > A mutation was reported to reduce transcription factor binding ability and transcription initiation activity and negatively control the gene transcription of LAMA1, playing a crucial role in pathological myopia [29]. In a study on degenerative diseases, D'Aoust and colleagues were the frst to discover that LAMA1 rs73938538 A > C was positively related to Alzheimer's disease in the Amish community [30]. Due to the single-nucleotide polymorphism of LAMA1, the mutated site seems unable to efectively translate LAMA1 into laminin subunit α1 so as to exert its specifc biological function, hence preventing esophageal cancer susceptibility. We observed that the consequences of two SNP loci that were related to esophageal susceptibility were stop-gain mutations, while the rest were synonymous and missense variants. Tis is largely in accordance with our expectations, especially when terminal gain mutations and missense variants can dramatically alter protein function, even with single nucleotide changes. However, we also reveal that in the ESCC population, LAMA1 polymorphisms did not show a statistically signifcant association with the degree of diferentiation, lymph node positivity, or T stage.

Journal of Oncology
Te main limitation of our study is that we only included populations from a specifc region in eastern China, which may result in a certain geographical specifcity and may not be generalized to the entire ESCC population. In addition, our study lacks replication in independent cohorts. Furthermore, we only discovered SNP sites in peripheral blood in our investigation. How these SNPs of LAMA1 translate into biological function in the evolution of esophageal carcinoma is defnitely a primary subject of our future investigation, which is currently technically difcult due to the lack of biological tools in our lab.
In conclusion, we found a strong association of LAMA1 rs62081531, rs539713, rs566655, and rs607230 polymorphisms with esophageal cancer susceptibility in the Chinese population. LAMA1 SNPs may signifcantly impact

Data Availability
Statistical results of this study are available from corresponding authors upon reasonable request. Specifc patient clinical information and genetic data to support the study results have not yet been made available due to the National BioSafety Law of the People's Republic of China. Table S1: Stratifed analyses between rs62081531 G > A polymorphism and ESCC risk by age, gender, smoking status, and alcohol consumption. Table S2: Stratifed analyses between rs621993 G > A polymorphism and ESCC risk by age, gender, smoking status, and alcohol consumption. Table S3: Stratifed analyses between rs539713 A > G polymorphism and ESCC risk by age, gender, smoking status, and alcohol consumption. Table S4: Stratifed analyses between rs566655 T > G polymorphism and ESCC risk by age, gender, smoking status, and alcohol consumption. Table S5: Stratifed analyses between rs73938538 A > C polymorphism and ESCC risk by age, gender, smoking status, and alcohol consumption. Table S6: Stratifed analyses between rs607230 T > C polymorphism and ESCC risk by age, gender, smoking status, and alcohol consumption. Table S7a: Te linkage disequilibrium test of LAMA1 in the case and control groups. Table S7b: Te linkage disequilibrium test of LAMA1 in the case and control groups.